Direct visualization of RecA protein binding to and unwinding duplex DNA following the D-loop cycle.

The RecA protein of Escherichia coli will promote the plectonemic joining of a linear single-stranded DNA molecule with a homologous supertwisted double-stranded (ds) DNA molecule. As shown by others, this reaction is characterized by a single cycle of joint formation and dissociation, termed the D-loop cycle. The released DNA products appear by electron microscopy to be topologically identical to the reactant DNAs, yet a second cycle of joining is not observed. This implies that either the RecA protein-single-stranded DNA filament or the dsDNA must be altered during the pairing reaction such that further joint formation is inhibited. Shibata et al. (Shibata, T., DasGupta, C., Cunningham, R. P., Williams, J. K. G., Osber, L., and Radding, C. M. (1982) J. Biol. Chem. 256, 7565-7572) proposed that the dsDNA was inactivated due to the binding of RecA protein following the D-loop cycle, but were unable to describe the structure of the putative RecA-protein-dsDNA complex. Here we have extended those studies to show that if fresh dsDNA is added to a reaction mixture following completion of the D-loop cycle, joint formation is stimulated, but only with the freshly added dsDNA. Following completion of the D-loop cycle, a labile RecA protein-dsDNA complex, in which the dsDNA is partially unwound can be preserved by glutaraldehyde fixation and visualized by electron microscopy. This result provides direct evidence that the block to a second cycle of joining is due to the presence of RecA protein remaining bound to the released dsDNA.